It is believed that all wounds are colonized by microbes. If the microbes reach a level of clinical infection, their presence is believed to impair healing and may be a contributing factor to wound chronicity. It has recently been estimated that hospital-acquired (nosocomial) infections are the fourth-leading cause of death in the United States, affecting 2 million patients per year and causing over 100,000 annual deaths, with a total annual cost of over $30 billion. Staphylococcal species such as S. epidermidis and S. aureus are responsible for the majority of nosocomial infections; treatment of these infections is often made much more challenging by the tendency of staphylococci to form biofilms.
Recently researchers have proposed that it may not be planktonic but rather biofilm communities which contribute to wound chronicity. Biofilms are polymicrobial groupings of bacteria which are held together in an extracellular polymeric substance consisting of protein, DNA, and polysaccarhides and are not totally susceptible to antibiotic treatment. It has been shown that 60% of the chronic wounds tested contained biofilm. (James et al., Wound Repair Regen., 16(1):37-44, 2008.)
Biofilms are populations of bacteria or fungi growing attached to an inert or living surface. Mounting evidence has shown that biofilms constitute a significant threat to human health. The Public Health Service estimates that biofilms are responsible for more than 80% of bacterial infections in humans (National Institutes of Health, 1998 RFA#DE-98-006). Examples of diseases caused by biofilms include dental caries, periodontitis, cystic fibrosis pneumonia, native valve endocarditis, and otitis media (Costerton et al. Science 1999 284:1318-1322), as well as infection of various medical devices such as urinary catheters, mechanical heart valves, cardiac pacemakers, prosthetic joints, and contact lenses (Donlan, R. M. 2001 Emerging Infect. Dis. 7:277-281). Fungi also form biofilms of clinical significance, for example Candida infections. Biofilm infections afflict tens of millions of patients in the U.S. annually and require a significant expenditure of health care dollars (Costerton et al. Science 1999 284:1318-1322). Bacteria growing in biofilms exhibit increased resistance to antimicrobial agents and are nearly impossible to eradicate. New methods for treating biofilm infections are needed.
Bacterial biofilms are sources of contamination that are difficult to eliminate in a variety of industrial, environmental and clinical settings. Biofilms are polymer structures secreted by bacteria to protect bacteria from various environmental attacks, and thus result also in protection of the bacteria from disinfectants and antibiotics. Biofilms may be found on any environmental surface where sufficient moisture and nutrients are present. Bacterial biofilms are associated with many human and animal health and environmental problems. For instance, bacteria form biofilms on implanted medical devices, e.g., catheters, heart valves, joint replacements, and damaged tissue, such as the lungs of cystic fibrosis patients. Biofilms also contaminate surfaces such as water pipes and the like, and render also other industrial surfaces hard to disinfect.
Biofilm is commonly known as the primary cause of many diseases and infections in biology. Biofilms also play a detrimental role on many other non biological surfaces. These biofilms, which exists not only on biological surfaces but also on all manner of surfaces, can be defined as a diverse community of microorganisms. The microorganisms bind tightly to one another, in addition to the solid surface, by means of an extracellular matrix consisting of polymers of both host and microbial origin.
Biofilms, exhibit an open architecture. The open architecture, which consists of channels and voids, helps to achieve the flow of nutrients, waste products, metabolites, enzymes, and oxygen through the biofilm. Because of this structure, a variety of microbial organisms can make up biofilms, including both aerobic and anaerobic bacteria. The microbial composition of biofilms includes a multitude of species of bacteria, archaea, fungi and viruses, which all exist in a relatively stable environment called microbial homeostasis. Biofilms are responsible for many of the diseases common in the body including dental diseases, non healing wounds and sores. Biofilms also are the cause of undesirable body odor resulting from biofilms on the body surfaces. Further biofilms are common on many engineering surfaces, and lead to material erosion, and to subpar engineering performance of these surfaces.
Bacterial biofilms are ubiquitous in nature and are usually defined as matrix-enclosed bacterial populations which adhere to each other and/or to surfaces or interfaces. Bacterial biofilm formation is an extremely common phenomenon with a major economic impact in different industrial, medical and environmental fields. Biofilms can comprise a single species or multiple species and can form on a wide range of abiotic and biotic surfaces and interfaces. Although polymicrobial biofilms predominate in most situations single species biofilms can occur under certain circumstances and are an increasing problem on the surface of medical implants. Growth as a biofilm offers a number of significant advantages to the bacterium over planktonic growth not the least of which is the attachment to the surface that enables the bacterium to localize itself in a favorable environment. In polymicrobial biofilms metabolic activities can be integrated and the presence of a variety of species allows for greater flexibility in metabolic and catabolic activities as the ‘genome’ of the biofilm population increases with increasing species diversity. The Centers for. Disease Control and Prevention estimate that 65% of human bacterial infections involve biofilms. Biofilms often complicate treatment of chronic infections by protecting bacteria from the immune system, decreasing antibiotic efficacy and dispersing planktonic cells to distant sites that can promote re-infection. Bacterial cells within a biofilm have been shown to be up to 500 times more resistant to certain antimicrobial agents than planktonic cells which is achieved by a number of processes including, the slowing of penetration of some antimicrobial agents into the biofilm matrix, the slowing of the growth rate of bacteria in the deeper layers of the biofilm and the binding of some antimicrobial agents to extracellular polymers thereby reducing the effective concentration. In addition, microbial biofilms have been described as microbial landscapes, which have a topography that protects against shear stress whilst allowing mass transfer. Most importantly in the oral cavity failure to attach and grow as a biofilm will rapidly result in clearance.
The oral cavity is a fertile environment for the growth of bacteria with a range of hard and soft tissue surfaces that provide a variety of distinctly different microhabitats. The stability of oral microbial biofilms requires dynamic balances by a range of synergistic and antagonistic interactions among species and the environment they create. Minor adjustments in the oral environment can affect these natural balances potentially leading to shifts in the ecology and changes in the species composition of oral microbial biofilms. For example, increased dental caries incidence is often caused by increased consumption of dietary carbohydrates, which is linked to the acidification of fluids at the tooth surface due to the bacterial fermentation of these carbohydrates. Experts agree that most forms of periodontal disease are caused by specific pathogens, particularly gram-negative bacteria. The microbial composition of dental biofilms includes over 700 species of bacteria and archaea, which all exist in a relatively stable environment called microbial homeostasis. (Kroes I, Lepp P W, Reiman D A Bacterial diversity within the human subgingival crevice. Proc Natl Acad Sci USA 1999; 96(25):14547-14552.)
Bacterial biofilms develop in variety of bodily cavities, including those of the ear, such as the middle ear, and of the nose, such as the frontal or maxillary sinuses, for example. Once bacterial growth has been established, the bacteria will often aggregate, stop dividing, and begin forming protective bacterial biofilm layers, or “slime layers,” comprised of polysaccharide matrices.
The protective bacterial biofilm interferes with the body's natural immune response as well as traditional methods of treatment. In particular, the bacteria emit exotoxins, which incite the body's immune system to respond with white cells. However, the bacterial biofilm interferes with the efficacy of the white cells' ability to attack the bacteria. The biofilm can also act as a barrier against topical administration of antibiotics and other medicaments.
Biofilm-forming bacteria also present obstacles to traditional, antibiotic treatments that act to kill dividing bacteria. In particular, the bacteria in a biofilm-forming state may have already ceased cell division, rendering such antibiotics largely ineffective. Antibiotic doses that kill free-floating bacteria, for example, need to be increased as much as 1,500 times to kill biofilm bacteria. At these high doses, the antibiotic is more likely to kill the patient before the biofilm bacteria. (Elder M J, at al. Biofilm-related infections in ophthalmology. Eye 1995; vol. 9 (Pt. 1):102-109.)
Methods of inhibiting biofilm formation in medical and industrial settings have previously been developed using metal chelators, specifically iron chelators. For example, U.S. Pat. No. 6,267,979, issued Jul. 31, 2001, to Raad et al., discloses the use of metal chelators in combination with antifungal or antibiotic compositions for the prevention of biofouling in water treatment, pulp and paper manufacturing and oil field water flooding. U.S. Pat. No. 7,314,857, issued Jan. 1, 2008, to Madhyastha, discloses synergistic antimicrobial compositions for inhibiting biofilm formation using combinations of an iron-sequestering glycoprotein, a cationic peptide, and an iron chelating agent. U.S. Pat. No. 7,446,089, issued Nov. 4, 2008, to Singh et al., is also directed to methods of inhibiting biofilm formation by limiting the amount of iron available to a population of bacteria, such that biofilm formation can be inhibited. These disclosures generally target iron, a higher affinity metal ion.
Given the serious medical, industrial, and environmental problems associated with bacterial biofilms, the need persists to develop targeted approaches to inhibit biofilm formation. Therefore, there it is desirable to develop an agent that efficiently controls and inhibits biofilm formation in medical and industrial applications.